- Email: [email protected]
Rapid and simple method for the determination of tryptophan in cereal grains
ANALYTICAL
BIOCHEMISTRY
Rapid
and
67, 206-2 19 ( 1975)
Simple
Method
of Tryptophan
for the Determination
in Cereal
JOSE MADRID
Grains1
CONCON
Department of Nutrition and Food Science, Unhaersity of Kentucky, Lexington, Kentucky 40506 Received December 25, 1974; accepted February 18, 1975 Alkaline extracts of cereal proteins are treated in the prescribed sequence: Glacial acetic acid-ferric chloride and 25.8 N H,SO, solutions. Specified amounts of these solutions and the sample are added in order to obtain the required sulfuric acid concentration in the reaction mixture. The reaction mixture is heated at 60°C for 45 min (cereal proteins) or 110 min (standard tryptophan). The absorbance of the resulting violet solution is read at 545 nm. Results are highly precise and agree quite favorably with those of the modified Spies and Chambers method using Pronase hydrolyzates. As little as 6-7 yg of tryptophan in cereal proteins may be determined reliably in samples with negligible blank corrections. The factors affecting color development and the accuracy and precision of the method are discussed.
Tryptophan is frequently a second limiting amino acid in cereal grains. It is also quite labile, especially in peptide form, in the presence of light, hydrogen ions, or less readily in the presence of hydroxyl ions. This destruction is, of course, accelerated by heat. Thus, tryptophan is usually not determined with the rest of the amino acids by the common chromatographic methods that require preliminary acid or even base hydrolysis at high temperatures. Modifications of the hydrolytic conditions have been proposed to prevent or minimize the destruction of tryptophan (l-3). Unfortunately, none of these modifications is effective in preventing significant destruction of the amino acid during chemical hydrolysis in the presence of large amounts of carbohydrates, such as in cereal grains (2). A number of spectrophotometric methods without preliminary chemical hydrolysis have been developed for the determination of tryptophan in proteins. A review of the various methods is given by Friedman and Finley (4). The procedure developed by Spies and Chambers (5,6), 1 Supported by Contract No. 12-14-100-9507 (71) from USDA Agricultural Research Service, Northern Utilization Research and Development Division, Peoria, IL 61604, and by the Department of Nutrition and Food Science, University of Kentucky, Lexington, KY 40506. Mention of firm names or trade products does not imply endorsement by USDA or the Department of Nutrition and Food Science.
206 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
RAPID
TRYPTOPHAN
METHOD
FOR
CEREALS
207
which has been modified and elaborated in subsequent papers (7-IO), is the most widely used. When the protein sample is first digested with proteolytic enzymes, such as Pronase (9,lO) or papain (1 l), the Spies and Chambers method appears to give accurate results even with crude protein preparations. A variation of the method was used with several food products by Lombard and Delange (12), with apparently good results. The Spies and Chambers method, however, is quite time consuming (consider: 4%hr enzyme hydrolysis: 6-18-hr reaction with the chromogen: OS-hr reaction with sodium nitrite). In addition, ethanol apparently interferes with the color development with p-dimethylaminobenzaldehyde (13) so that corn proteins extracted by the technique of Concon (14) cannot be used with this method. A rapid, simple and very sensitive method was reported by Fischl (15) using acetic acid as the chromogen. This method is actually based on the Adamkiewicz (16) reaction, since glyoxylic acid is usually present as an impurity in glacial acetic acid preparations. The uncertainty as to the presence of this impurity in each batch of glacial acetic acid prompted Opienska-Blauth and co-workers ( 17) to add small amounts of ferric chloride to glacial acetic acid in order to generate glyoxylic acid in the presence of sulfuric acid. 1 have attempted to use the method of Opienska-Blauth and coworkers ( 17) on unhydrolyzed cereal protein extracts. Unfortunately, several of the samples gave serious browning and other interfering color reactions. Furthermore, the values obtained, even without browning, generally differed considerably from those of the Spies and Chambers method (6,9,10). The present paper presents a rapid, sensitive and simple method for the determination of tryptophan in cereal grains without enzyme hydrolysis. It utilizes the reagent of Opienska-Blauth and co-workers (17) but under conditions which do not result in browning and other interfering color reactions. The values obtained compare favorably with those of the Spies and Chambers method with samples hydrolyzed with Pronase (9,lO). MATERIALS
AND
METHODS
(Refer to Notes following the Materials and Methods ther elaboration on the various items that follow). Reagents
and Standard
section for fur-
Solutions
A. Glacial acetic acid-ferric chloride solution (I 7). Dissolve 5.4 g of FeCl,v6H,O in 10 ml of water containing a few drops of acetic acid to prevent formation of insoluble Fe(OH),. To 0.5 ml of this solution, add
208
JOSE
MADRID
CONCON
glacial acetic acid in a volumetric flask to make 1 liter. Mix. Store in brown bottle. This reagent is stable indefinitely. B. Sulfuric acid. 25.8 N. C. Miscellaneous solutions. NaOH, 0.075 and 0.125 N; ethanol, 70%. Preferably when large numbers of samples are involved, all the preceding solutions are dispensed from accurate automatic or semiautomatic dispensers (for example, Digital Dispensers or Repipet, ColeParmer Instrument Co. Chicago, IL 60648). D. Standard tryptophan solution. Dissolve about 20.00 mg of L-tryptophan (corrected for moisture content) in about 30 ml of 0.075 N NaOH solution in a 500-ml volumetric flask. Make up to the mark with water. Mix. This solution is stable if kept refrigerated in a dark bottle (Note 1). Preparation
of Samples
All cereal grains with loose hulls may be dehulled rapidly and conveniently by a technique described previously by Concon (18). All are ground to 150 mesh, except floury corns which are ball milled to 200 mesh (14). A grinding apparatus, such as the water-cooled Analytical Mill (National Scientific Co., Cleveland, OH 44146) which prevents exposure of sample to excessive heat during the grinding process is preferred (Note 2). Highly pigmented whole grain samples (for example, rye, barley, oats, and red wheat) which give unusually high blanks are depigmented as follows (Note 3): Cover a chosen weight of powdered sample with Nbutanol half-saturated with water (10 ml of water/ 100 ml of butanol). Vortex mix vigorously for a few minutes. Centrifuge for a few minutes at 5,000 rpm and decant. Repeat butanol treatment until the supernatant fluid is only slightly colored. Likewise, extract the residue from the butanol treatment, successively, with an anhydrous ether-absolute ethanol mixture (1: 1, v/v) and hexane or petroleum ether. Loosen the packed residue after the last extraction with a glass rod and dry in a vacuum desiccator by continuous application of a vacuum pump or water aspirator (Note 4). Sieve (150 mesh) the dry material and store in air-tight bottles in the refrigerator. Determine the percent nitrogen of the powder using the rapid microKjeldahl procedure (19). Protein
Extraction
All samples, except corn, are extracted as follows: Pipet into a 15-ml heavy-duty polypropylene test tube with corresponding closure, 5 ml of
RAPID
TRYPTOPHAN
METHOD
FOR
CEREALS
209
0.075 N NaOH solution (Note 5). With a torsion balance accurate to 10 mg (for example, Mettler P1200N, Mettler Instrument Corp., Princeton, NJ 08540), weigh about 100-200 mg of sample (Note 5). Transfer to the polypropylene tube containing the NaOH solution. Cover tightly with the appropriate closure. Vortex mix vigorously until no clumps are evident, and continue mixing for an additional 4-5 min. Centrifuge at 8,000 r-pm for 10 min. Carefully shake back into the supernatant fluid without disturbing the sediments, any creamy mass (lipoproteins) which accumulates on the surface. Transfer to a clean test tube (Note 5). Corn samples are extracted as described previously by Concon (13). Determine the total nitrogen in 1 ml of extract using the rapid microKjeldahl procedure (19) (Note 6). Tryptophan Determination 1. Pipet directly into the bottom of a 15-ml test tube with a screw cap, 1.00 ml of protein extract containing 20-50 pg of tryptophan (Notes 5 and 7). For the reagent blank, pipet 1 ml of 0.075 N NaOH solution instead of protein extract. Pipet 1.00 ml of standard tryptophan solution (Solution D, Reagents and Standard Solutions) into a separate test tube. 2. Add 3.0 ml of glacial acetic acid-ferric chloride solution. Vortex mix. Add this solution first to all samples before proceeding to Step 3 (Note 8). 3. Add 2.0 ml of 25.8 N sulfuric acid. Vortex mix to a homogeneous solution immediately after adding the acid. Cover loosely with a screw cap (Note 8). 4. Heat all test solutions simultaneously for 45 min at 60°C in a constant-temperature water bath which is shielded from direct laboratory light. The standard tryptophan solution is heated for 110 min (Note 9). 5. After the heating period, cool to room temperature in an ice-water bath for l-2 min and read the absorbance against the reagent blank at 545 nm (Beckman DU). Preferably, use a spectrophotometer flow cell when large number of samples are determined (Note 10). 6. In general, a sample blank should be determined. Proceed as follows: Add to 1.00 ml of protein extract the same reagents as in steps 2 and 3. Likewise, include a reagent blank. Cool in an ice-water bath each time a reagent is added. Vortex mix thoroughly after adding sulfuric acid solution, and, while the sample is in the ice-water bath, apply suction (water aspirator) cautiously to remove bubbles which interfere in the absorbance readings. Break the suction when a foam threatens to escape out. Read the absorbance at 545 nm against the reagent blank (unheated) within 4-5 min after addition of acid (Note 10). 7. Calculations (Note 11).
210
JOSE
MADRID
CONCON
T= ((4--A,,) x VIA’, F= (T,,c,M,d x 16, where, T = of sample: tryptophan solution: N
[II [2] 2
gram(s) of tryptophan in 100 gm protein’: A, = absorbance Ash = absorbance of sample blank; Astd = absorbance of tryptophan in 1 ml standard standard; Tstd = milligram(s) = milligram(s) nitrogen in 1 ml of protein extract.
Notes 1. Tryptophan dissolves more rapidly in alkaline or acid solution. However, it is more stable in slightly alkaline medium. Recrystallize impure tryptophan twice from boiling water (5 g/200 ml). Collect the crystals in a sintered-glass funnel and wash with absolute ethanol each time. Remove residual alcohol at 80°C (forced-draft oven) and store over concentrated H,SO, in vacua. Determine residual moisture on an aliquot by drying at 110°C. 2. Loss of tryptophan in the samples by oxidation or photolysis on prolonged storage, especially in the presence of moisture, may be minimized by refrigeration in air-tight brown bottles. Peptide-bound tryptophan, being more reactive (see Results and Discussion) may be more susceptible to destruction. Loss of tryptophan presumably is minimized during grinding by water-cooling. 3. Proper choice of reaction conditions and butanol extraction of sample minimize, if not eliminate the interference of grain pigments that posed serious difficulties with the Opienska-Blauth method (17). 4. To prevent scattering and contamination of samples, cover them with several layers of tissue paper tied securely to the tube before drying the sample with a vacuum pump. 5. The torsion balance is ideal for rapid weighing of several samples for extraction. Time is saved if one sample is extracted while the next one is being weighed. Accurate weighing is not necessary since the nitrogen of the extract is determined. Formation of clumps which are difficult to disperse is prevented by adding the sample to the NaOH solution instead of the reverse. The recommended weights of sample correspond to between 20 and 40 ,ug of tryptophan. The maximum tryptophan value which still conforms to Beer and Lambert’s law does not exceed 60 pg (Results and Discussion). Between 250 and 300 mg may be required for the relaZThe follows:
Jones (20) nitrogen-protein conversion factors (g of N/l00 g of protein) Barley, 17.2; corn, 16.0: oats, 17.2: rice, 16.8: rye, 17.1; wheat 17.1.
are as
RAPID
TRYPTOPHAN
METHOD
FOR
CEREALS
211
tively low-tryptophan grains, such as corn and barley. Interferences are minimized with smaller sample weights. 6. With 0.075 N NaOH, the protein extraction efficiency, of samples stored in the refrigerator for several months is 90% or greater. The value for freshly ground samples generally is 98% or greater. 7. Poor reproducibility of results may be obtained if the extract is allowed to flow down the side of the test tube during pipetting. The reason is that an indefinite but significant amount of protein may remain on the side of the tube which, obviously, may not be reacted equally with the rest of the sample, if at all. 8. Serious browning or even charring may result if H,SO, is added first to the sample, instead of acetic acid. The large amount of heat produced when H&SO, is added to the alkaline extract may destroy most of the tryptophan. 9. Proceed to determine the absorbance of the samples while the standard solution is still developing in the water bath. For each batch of reagents, include a tryptophan standard for each daily run (see Note 11). For precision, immerse at least 3/4 of the tube during heating to insure equal heating conditions for any liquid on the side of the tube. 10. Factors such as inherent sample turbidity, which is not removed by filtration or centrifugation, may result in still significant blank values in spite of butanol extraction. When extracts are clear and almost colorless, blank corrections may be omitted. Even though some samples may develop color at 25°C faster (e.g., wheat) than others, the time lag of 5-6 min, before a significant absorbance due to the chromophore can be detected, permits the determination of the sample blank. Samples are cooled in ice water to delay color development during removal of bubbles. However, readings must be done with samples at almost room temperature. Only one sample blank at a time may be processed for absorbance reading within 5 min without use of a flow cell. Otherwise, 2-3 samples are possible. Since the test solutions are more viscous than water, bubbles can only be effectively removed by suction or by heating. Very small bubbles which are invisible may coalesce during absorbance reading. No bubbles are present in the test solutions heated at 60°C. Thus, the unheated sample blanks must be suctioned thoroughly to insure complete removal of bubbles. 11. A standard curve may be plotted with each fresh batch of reagents. However, it is preferable to include a standard tryptophan solution with each run as an internal check for possible variation in concentrations of reagents or reaction conditions (see Results and Discussion).
212
JOSE
MADRID
CONCON
TABLE TRYPTOPHAN
CONTENT
SPECIES
OF CEREAL AND
Cereal grams Barley Missouri B 415 Knob CI 11910 McNair 601 Corn Tolima 377 Brazil 70 Opaque-2 Oats Commercial Mix Wyndmere Rice BPI-76- 1d IR-8 LPd IR-8 HP@ Wheat Hard red Winter wheat Azteca 67 Unbleached Floure Rye Tetra Petcus Michigan rye
THE
OF PROTEINS GRAINS PROCEDURE
1
FROM
DIFFERENT
AS OBTAINED OF SPIES
WITH AND
VARIETIES THE
PRESENT
OF VARIOUS METHOD
(6.9)
CHAMBERS
Tryptophan, g/l00 g of proteins
Total nitrogen (%I”
Total nitrogen extracted m
Present methodD,C
Spies and ChambersbSc
1.63 2.40 2.47
98.3 100.7 97.8
1.71 1.39 1.49
1.45 1.47 1.29
1.79 1.12 1.11
92.22 100.7 105.6
0.41 0.52 0.88
0.32 0.47 0.75
2.49 2.29
100.0 99.6
1.02 1.20
1.00 1.22
2.52 1.19 1.62
97.5 90.0 88.3
1.42 1.44 I .59
1.42 1.36 1.43
2.89 2.88
97.7 99.6
1.10 1.18
1.04 1.36
2.27
103.6
0.86
0.81
1.79 2.17
95.8 92.9
0.99 I .03
0.83 0.85
u “As-is” basis. b Average of duplicate determinations. Average difference of individual values from the corresponding mean is 0.01. c Nitrogen-protein conversion factor: 16 g of N/100 g of protein, except rice for which 16.8 g was used. d Samples supplied by Dr. B. Juliano, International Rice Research Institute, Los Baiios, Laguna, Philippines. p Unknown variety.
RESULTS AND
DISCUSSION
The tryptophan content of the proteins of several varieties of different species of cereal grains are shown in Table 1. The values obtained with this method are compared with those of the method of Spies and Chambers (5,6,9). Agreement between the two methods is highly satis-
RAPID
TRYPTOPHAN
METHOD
FOR
CEREALS
213
factory. Values recorded in Table 1 are very precise. The average difference between the mean and the corresponding individual values of each duplicate is about 0.03 units. Except for corn, samples analyzed with the Spies and Chambers method were first extracted with 0.075 N NaOH. The extract (0.50 ml) was treated with 0.5 ml of Pronase solution [4 mg of PronaselO.5 ml of phosphate buffer, 0.1 M, pH 4.4 (Pronase activity, 45,000 PUK units/g, Calbiochem, Los Angeles, CA 90054)], and digested for 48 hr at 40°C with a few drops of toluene as preservative. The final mixture had a pH between 7.5 and 8.5, the optimum range for Pronase activity (9,21). Ethanol interference (13) precluded the use of the extraction technique of Concon (14) for corn proteins in conjunction with the Spies and Chambers (5,6,9) method. Instead, corn powder was digested directly with Pronase for 48 hr at 4O”C, based on the recommendation of Spies (9). [About 100 mg of corn/ml of Pronase solution (2 mg of crude enzyme/ml of 0.1 M phosphate buffer, pH 7.5).] Between 93 and 103% of corn nitrogen was solubilized; the digest did not give any abnormal colors with the reagents. For the sample blank, NaNO, was added first to the sample before the p-dimethylaminobenzaldehyde (DAB)-H,SO, solution. The absorbance was read immediately. Many samples developed between 20 and 40% of the maximum color intensity obtainable in the oxidation with NaNO, during the 18-hr incubation with DAB and H&SO,. This suggests the presence of unspecified oxidants in cereal grains. Conformance
to Beer and Lambert’s
Law
As shown in Fig. 1, the glyoxylate reaction under the conditions in this method conforms very well to the Beer and Lambert’s law only up to about 60 pg of free or cereal protein-bound tryptophan. There is a gradual deviation above this value so that Eq. [l] is no longer valid. The deviation appears to be a property of the glyoxylate reaction and not an indication of the exhaustion of the available glyoxylate since, from 20- 100 pg of tryptophan, the glyoxylate reaction under the conditions in this method can be more precisely represented by Eq. [ 31 instead of the Beer and Lambert’s law. Log T = k/l + log b,
[31
where T = tryptophan concentration; A = absorbance; k = a proportionality constant, and b = the tryptophan value at zero absorbance, in this case between 11 and 12 pg. The lack of correlation of the experimental results below 20 pg in this case may be ascribed to experimental error which is greatly exaggerated by a logarithmic plot. Thus, Eq. 3 may be used for calculating tryptophan values between 20 and 100 pg.
214
JOSE MADRID
CONCON
.240 s 2 2 8 z
.200
0
CORN
-O-
OATS
0
W"EA1P
-160
TRYPTOPHAN
,120 ,120
.OPO
.040 .020 10
20
40
60 ),g
80
100
120
TRYPTOPHAN
FIG. 1. A Beer and Lambert’s plot of the glyoxylate reaction with tryptophan and various unhydrolyzed cereal proteins.
But because of its simplicity, calculations based on the Beer and Lambert’s law (Eq. [l] is preferable for tryptophan values below 60 pg. Sensitivity, Accuracy and Precision, and Specificity of the Method The minimum amount of tryptophan that may be determined reliably in this method is about 6-7 pg (Fig. l), provided the sample blank is negligible. An ultramicroadaptation would lower this value to at least 1 t-a* The accuracy of this method may be compared only with the Spies and Chambers method (6,9). The highly satisfactory agreement of the results obtained with these methods has already been mentioned. A possible source of error in the Spies and Chambers method (6,9) is the destruction of tryptophan during the prolonged incubation in 19 N
RAPID
TRYPTOPHAN
METHOD
FOR
CEREALS
215
H,SO, (22). However, free tryptophan was shown to be remarkably stable in 19 N H,SO, (5), and Pronase digestion, very likely, liberates all the tryptophan residues in cereal proteins (20). The Spies and Chambers method has been used satisfactorily with various food products (12,23), with tryptophan values in satisfactory agreement with the results of microbiological assays (23). The slight browning of solutions observed during incubation with H,SO, is minimized if not eliminated by NaNO,. This browning may be a source of positive error in the Spies and Chambers method. The high precision of the present method has already been mentioned. Individual results for the same sample obtained on different days differed by no more than 0.03 units. Other indoles which also react with glyoxylate in the presence of H,SO, (5,24) are not present in cereal grain extracts in significant amounts, if at all. No other compounds in cereal grains are known to react significantly with glyoxylate. And unlike in the Spies and Chambers method (5,6,9), ethanol does not interfere. With the sample blank correction, the present method is highly specific for tryptophan in cereal grains. The Tryptophan Chromophore and Its Stability Normally, the tryptophan chromophore gives a violet solution in acid medium. But occasionally, as in rye, barley, and, sometimes, wheat, the solution is red-violet or magenta, without affecting the A,,,. The reddish hue of the solution, in this case, may be due to certain grain pigments with negligible absorption at 545 nm. The color of the solution is stable even after several hours in bright laboratory light. However, light may have a slight (about 4%) colordeepening effect during development. A similar effect of light was also observed by Spies and Chambers (7) with DAB. Thus, because of possible unequal illumination, the samples were covered during color development for maximum precision. The tryptophan chromophore is stable even after 1 hr at 60°C. Only one barley sample showed a slight decrease within this time. However, above 70°C the chromophore is gradually destroyed at different rates in different samples. Barley and corn showed faster rates of destruction than the other grains. And at 100°C 80% or more of the chromophore in most samples may be destroyed in 10 min. Effects of Sulfuric Acid and Water Concentrations As seen in the following schemes, H,SO, functions both as a proton donor and dehydrating agent in the formation of the tryptophan chromophore (Scheme I) and of glyoxylate from acetic acid (Scheme II).
216
JOSE MADRID Scheme
I
[Formation
mtyg Scheme
II
of
the
Tryptophan
(Formation
(4,25)1
of
2
OOH
Glyoxylate)
H2S04
*
) (01,
Chromophore
*cg-;ooH ;;-TTH < ,H--
Fe +++ CH3COOH
CONCON
H+
<-
(OH)2-CH-COOH glyoxylic hemihydrate
O=CH-COOH S20
acid
glyoxylic
acid
In cereal proteins (represented by rice and wheat), color development, expressed as percent of the maximum color obtained under the conditions chosen for this method, is a direct and inverse function of the H,SO, and H,O concentrations, respectively (Fig. 2A and B). For the data in Fig. 2, a IO-min reaction time was chosen since differences in reaction rates are more pronounced in the initial stages of the reaction. 160
160
4
6
E
10
12
NORMALITY OF H2S04 IN THE FINAL MIXTURE
MOLARITY OF II20 IN THE FINAL MIXT”,?Z?
FIG. 2. Effect of (A) H,SO, and (B) H,O concentrations on the color development of the glyoxylate reaction with free and cereal protein-bound tryptophan.
RAPID
TRYPTOPHAN
METHOD
FOR
CEREALS
217
The steep slope of these curves (3.5) represents a 17% increase in color intensity for each 1 N increment in acid concentration. Thus, color development is highly sensitive to acid concentration. The color development of free tryptophan under the same conditions as with protein samples gives a sigmoid-shaped curve (Fig. 2) which can be expressed semilogarithmically as in Eq. [4]. LogA=bS-CC,
[41
where A = absorbance, S = acid or water concentration, C = the concentration at zero absorbance; and b = slope of the curve which is negative when S is the water concentration. The sigmoid curve, in contrast to the linear cereal-protein curves, suggests the possible influence of the free amino or carboxylic groups in the color development. Experimentally, Eq. [4] is valid only up to 10 N H&SO,, since, above this value, significant destruction of tryptophan occurs which is accompanied by increased browning, in the case of the cereal extracts, or increased cloudiness, in the case of the standard. Thus, 10 N H,SO, is the practical limit to which acid concentration can be increased for the purpose of increasing the sensitivity of the method. In rye and barley, browning and other-color formations occur even at a much lower acid concentration. [These grains produced red solutions in the OpienskaBlauth method ( 17)]. The primary function of H,SO, in the reaction is probably as a dehydrating agent rather than as a proton donor. This function is probably aptly illustrated in Fig. 2B which shows a rapid decrease in color with increasing water concentration, and a zero absorbance at 20 M H,O. The sensitivity of the glyoxylate reaction to water concentration was also observed previously by others (15, 17, 22). The dehydration of the intermediate carbinol in Scheme I may be influenced by the water concentration, not only by mass action but also by its effect on the dehydrating capacity of H,SO,. In addition, increased water concentration may also favor the formation of a very stable glyoxylate hemihydrate (26) which is expected to be less reactive to tryptophan. The Rate of Color Formation
In Fig. 3, one notes that, between 60 and 70°C only acid concentration among the factors studied can affect the time maximum color is attained with free tryptophan. Higher temperatures cannot be tested because of the destruction of tryptophan or its chromophore. Copper, whose color-enhancing effect was noted by Winkler (27), has no effect on the time of maximum color development. On the contrary, its use in
218
JOSE
MADRID
CONCON
.22,
.20.
&iYTrzlo4, 7o”c
.18
Pd ,
.I6
.
8.6
N i12S04,
GOOC +
COPPER
-
d II
0
f3.G N H2S”4,
60°C
0
7.9
GODC
/cfi
.14 _
El H7S04,
e 2Fj
. 12.
5 .10.-
.08 -
.OG _
.04
-
MINUTES
FIG. 3. The rate of color development of the glyoxylate reaction with free tryptophan under different conditions. Inset: The rate of color development of the glyoxylate reaction with different cereal proteins at 60°C.
the present method is precluded since it promotes intense browning of solutions in proportion to the amount of metal present. Among the cereal grains (Fig. 3, inset), only oats show a slight delay in the attainment of maximum color, which, for most grains, occurs after 30 min at 60°C. ACKNOWLEDGMENTS I thank Dr. R. J. Dimler, Northern Regional Research Laboratory, USDA, Peoria, IL; Dr. E. L. Kendrick, Cereal Crops Research Branch, Plant Industry Station, USDA, Beltsville, MD; and Dr. L. V. Packett, Department of Nutrition and Food Science, University of Kentucky, Lexington, KY 40506, for making this work possible; and especially Mr. C. H. Van Etten, Northern Regional Research Laboratory, Peoria, IL, for his valuable assistance and for providing the samples: I also thank many generous individuals for their efforts in providing most of the samples and others, for their technical and secretarial assis-
RAPID
TRYPTOPHAN
METHOD
FOR
CEREALS
219
tance. My thanks also to Mr. Jerry Reisig for his inadvertent assistance which helped in speeding up the work.
REFERENCES 1. 2. 3. 4. 5. 6.
7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
Matsubara, H., and Sasaki, R. M. (1969) Biochem. Biophys. Res. Commun. 35, 175. Penke, B.. Ferenczi, R.. and Kovacs, K. (1974) Anal. Biochem. 60,45. Liu, T. Y., and Chang, Y. H. (1971) J. Biol. Chem. 246, 2842. Friedman, M., and Finley, J. W. ( 197 1) .I. Agr. Food Chem. 19, 626. Spies, J. R., and Chambers, D. C. (1948) Anal. Chem. 20, 30. Spies, J. R., and Chambers, D. C. ( 1949) Anal. Chem. 21, 1249. Spies, J. R., and Chambers, D. C. (1950) Anal. Chem. 22, 1209. Spies, J. R. (1950) Anal. Chem. 22, 1447. Spies, J. R. (1967) Anal. Chem. 39, 1412. Spies, J. R. (1968)5. Agr. Food Chem. 16, 514. Vangala, R. R., and Menden, E. (1970) Z. Lebensm. Untersuch. Forsch. 142, 195. Lombard, J. H., and Delange, D. J. (1965) Anal. Biochem. 10, 260. Concon, J. M. (1975) Anal. Biochem, in press. Concon, J. M. (1973) Anal. Biochem. 55, 563. Fischl, J. (1960) J. Biol. Chem. 235, 999. Adamkiewicz, A. (1875) Berichte 8, 1 11. Opienska-Blauth, J., Charenzinski. M., Berbec, H. (1963) Anal. Biochem. 6, 69. Concon, J. M. (1975) Anal. Biochem., in press. Concon, J. M., and Soltess. D. (1973) Anal. Biochem. 53, 35. Jones, D. B. (1931) U.S.D.A. Circular No. 183, Washington, DC. Nomoto, M.. Narahashi, Y., and Murakami, M. (1960) J. B&hem. (Tokyo) 48, 593. Inglis, A. S., and Leaver, I. H. (1964) Anal. Biochem. 7, 10. Food and Agricultural Organization of the United Nations (1970) Ammo Acid Contents of Foods and Biological Data on Proteins, Rome. Remers, W. A. (1972) in Indoles Part I (Houlihan, W. J., ed.), p. 1, Wiley-Interscience, New York. Lugg, J. W. H. (1938) Biochem. J. 32, 775. Fieser, L. T., and Fieser, M. (1956) Organic Chemistry, D. C. Heath, Boston. Winkler, S. (1934) Z. Physiol. Chem. 228, 50.
BIOCHEMISTRY
Rapid
and
67, 206-2 19 ( 1975)
Simple
Method
of Tryptophan
for the Determination
in Cereal
JOSE MADRID
Grains1
CONCON
Department of Nutrition and Food Science, Unhaersity of Kentucky, Lexington, Kentucky 40506 Received December 25, 1974; accepted February 18, 1975 Alkaline extracts of cereal proteins are treated in the prescribed sequence: Glacial acetic acid-ferric chloride and 25.8 N H,SO, solutions. Specified amounts of these solutions and the sample are added in order to obtain the required sulfuric acid concentration in the reaction mixture. The reaction mixture is heated at 60°C for 45 min (cereal proteins) or 110 min (standard tryptophan). The absorbance of the resulting violet solution is read at 545 nm. Results are highly precise and agree quite favorably with those of the modified Spies and Chambers method using Pronase hydrolyzates. As little as 6-7 yg of tryptophan in cereal proteins may be determined reliably in samples with negligible blank corrections. The factors affecting color development and the accuracy and precision of the method are discussed.
Tryptophan is frequently a second limiting amino acid in cereal grains. It is also quite labile, especially in peptide form, in the presence of light, hydrogen ions, or less readily in the presence of hydroxyl ions. This destruction is, of course, accelerated by heat. Thus, tryptophan is usually not determined with the rest of the amino acids by the common chromatographic methods that require preliminary acid or even base hydrolysis at high temperatures. Modifications of the hydrolytic conditions have been proposed to prevent or minimize the destruction of tryptophan (l-3). Unfortunately, none of these modifications is effective in preventing significant destruction of the amino acid during chemical hydrolysis in the presence of large amounts of carbohydrates, such as in cereal grains (2). A number of spectrophotometric methods without preliminary chemical hydrolysis have been developed for the determination of tryptophan in proteins. A review of the various methods is given by Friedman and Finley (4). The procedure developed by Spies and Chambers (5,6), 1 Supported by Contract No. 12-14-100-9507 (71) from USDA Agricultural Research Service, Northern Utilization Research and Development Division, Peoria, IL 61604, and by the Department of Nutrition and Food Science, University of Kentucky, Lexington, KY 40506. Mention of firm names or trade products does not imply endorsement by USDA or the Department of Nutrition and Food Science.
206 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
RAPID
TRYPTOPHAN
METHOD
FOR
CEREALS
207
which has been modified and elaborated in subsequent papers (7-IO), is the most widely used. When the protein sample is first digested with proteolytic enzymes, such as Pronase (9,lO) or papain (1 l), the Spies and Chambers method appears to give accurate results even with crude protein preparations. A variation of the method was used with several food products by Lombard and Delange (12), with apparently good results. The Spies and Chambers method, however, is quite time consuming (consider: 4%hr enzyme hydrolysis: 6-18-hr reaction with the chromogen: OS-hr reaction with sodium nitrite). In addition, ethanol apparently interferes with the color development with p-dimethylaminobenzaldehyde (13) so that corn proteins extracted by the technique of Concon (14) cannot be used with this method. A rapid, simple and very sensitive method was reported by Fischl (15) using acetic acid as the chromogen. This method is actually based on the Adamkiewicz (16) reaction, since glyoxylic acid is usually present as an impurity in glacial acetic acid preparations. The uncertainty as to the presence of this impurity in each batch of glacial acetic acid prompted Opienska-Blauth and co-workers ( 17) to add small amounts of ferric chloride to glacial acetic acid in order to generate glyoxylic acid in the presence of sulfuric acid. 1 have attempted to use the method of Opienska-Blauth and coworkers ( 17) on unhydrolyzed cereal protein extracts. Unfortunately, several of the samples gave serious browning and other interfering color reactions. Furthermore, the values obtained, even without browning, generally differed considerably from those of the Spies and Chambers method (6,9,10). The present paper presents a rapid, sensitive and simple method for the determination of tryptophan in cereal grains without enzyme hydrolysis. It utilizes the reagent of Opienska-Blauth and co-workers (17) but under conditions which do not result in browning and other interfering color reactions. The values obtained compare favorably with those of the Spies and Chambers method with samples hydrolyzed with Pronase (9,lO). MATERIALS
AND
METHODS
(Refer to Notes following the Materials and Methods ther elaboration on the various items that follow). Reagents
and Standard
section for fur-
Solutions
A. Glacial acetic acid-ferric chloride solution (I 7). Dissolve 5.4 g of FeCl,v6H,O in 10 ml of water containing a few drops of acetic acid to prevent formation of insoluble Fe(OH),. To 0.5 ml of this solution, add
208
JOSE
MADRID
CONCON
glacial acetic acid in a volumetric flask to make 1 liter. Mix. Store in brown bottle. This reagent is stable indefinitely. B. Sulfuric acid. 25.8 N. C. Miscellaneous solutions. NaOH, 0.075 and 0.125 N; ethanol, 70%. Preferably when large numbers of samples are involved, all the preceding solutions are dispensed from accurate automatic or semiautomatic dispensers (for example, Digital Dispensers or Repipet, ColeParmer Instrument Co. Chicago, IL 60648). D. Standard tryptophan solution. Dissolve about 20.00 mg of L-tryptophan (corrected for moisture content) in about 30 ml of 0.075 N NaOH solution in a 500-ml volumetric flask. Make up to the mark with water. Mix. This solution is stable if kept refrigerated in a dark bottle (Note 1). Preparation
of Samples
All cereal grains with loose hulls may be dehulled rapidly and conveniently by a technique described previously by Concon (18). All are ground to 150 mesh, except floury corns which are ball milled to 200 mesh (14). A grinding apparatus, such as the water-cooled Analytical Mill (National Scientific Co., Cleveland, OH 44146) which prevents exposure of sample to excessive heat during the grinding process is preferred (Note 2). Highly pigmented whole grain samples (for example, rye, barley, oats, and red wheat) which give unusually high blanks are depigmented as follows (Note 3): Cover a chosen weight of powdered sample with Nbutanol half-saturated with water (10 ml of water/ 100 ml of butanol). Vortex mix vigorously for a few minutes. Centrifuge for a few minutes at 5,000 rpm and decant. Repeat butanol treatment until the supernatant fluid is only slightly colored. Likewise, extract the residue from the butanol treatment, successively, with an anhydrous ether-absolute ethanol mixture (1: 1, v/v) and hexane or petroleum ether. Loosen the packed residue after the last extraction with a glass rod and dry in a vacuum desiccator by continuous application of a vacuum pump or water aspirator (Note 4). Sieve (150 mesh) the dry material and store in air-tight bottles in the refrigerator. Determine the percent nitrogen of the powder using the rapid microKjeldahl procedure (19). Protein
Extraction
All samples, except corn, are extracted as follows: Pipet into a 15-ml heavy-duty polypropylene test tube with corresponding closure, 5 ml of
RAPID
TRYPTOPHAN
METHOD
FOR
CEREALS
209
0.075 N NaOH solution (Note 5). With a torsion balance accurate to 10 mg (for example, Mettler P1200N, Mettler Instrument Corp., Princeton, NJ 08540), weigh about 100-200 mg of sample (Note 5). Transfer to the polypropylene tube containing the NaOH solution. Cover tightly with the appropriate closure. Vortex mix vigorously until no clumps are evident, and continue mixing for an additional 4-5 min. Centrifuge at 8,000 r-pm for 10 min. Carefully shake back into the supernatant fluid without disturbing the sediments, any creamy mass (lipoproteins) which accumulates on the surface. Transfer to a clean test tube (Note 5). Corn samples are extracted as described previously by Concon (13). Determine the total nitrogen in 1 ml of extract using the rapid microKjeldahl procedure (19) (Note 6). Tryptophan Determination 1. Pipet directly into the bottom of a 15-ml test tube with a screw cap, 1.00 ml of protein extract containing 20-50 pg of tryptophan (Notes 5 and 7). For the reagent blank, pipet 1 ml of 0.075 N NaOH solution instead of protein extract. Pipet 1.00 ml of standard tryptophan solution (Solution D, Reagents and Standard Solutions) into a separate test tube. 2. Add 3.0 ml of glacial acetic acid-ferric chloride solution. Vortex mix. Add this solution first to all samples before proceeding to Step 3 (Note 8). 3. Add 2.0 ml of 25.8 N sulfuric acid. Vortex mix to a homogeneous solution immediately after adding the acid. Cover loosely with a screw cap (Note 8). 4. Heat all test solutions simultaneously for 45 min at 60°C in a constant-temperature water bath which is shielded from direct laboratory light. The standard tryptophan solution is heated for 110 min (Note 9). 5. After the heating period, cool to room temperature in an ice-water bath for l-2 min and read the absorbance against the reagent blank at 545 nm (Beckman DU). Preferably, use a spectrophotometer flow cell when large number of samples are determined (Note 10). 6. In general, a sample blank should be determined. Proceed as follows: Add to 1.00 ml of protein extract the same reagents as in steps 2 and 3. Likewise, include a reagent blank. Cool in an ice-water bath each time a reagent is added. Vortex mix thoroughly after adding sulfuric acid solution, and, while the sample is in the ice-water bath, apply suction (water aspirator) cautiously to remove bubbles which interfere in the absorbance readings. Break the suction when a foam threatens to escape out. Read the absorbance at 545 nm against the reagent blank (unheated) within 4-5 min after addition of acid (Note 10). 7. Calculations (Note 11).
210
JOSE
MADRID
CONCON
T= ((4--A,,) x VIA’, F= (T,,c,M,d x 16, where, T = of sample: tryptophan solution: N
[II [2] 2
gram(s) of tryptophan in 100 gm protein’: A, = absorbance Ash = absorbance of sample blank; Astd = absorbance of tryptophan in 1 ml standard standard; Tstd = milligram(s) = milligram(s) nitrogen in 1 ml of protein extract.
Notes 1. Tryptophan dissolves more rapidly in alkaline or acid solution. However, it is more stable in slightly alkaline medium. Recrystallize impure tryptophan twice from boiling water (5 g/200 ml). Collect the crystals in a sintered-glass funnel and wash with absolute ethanol each time. Remove residual alcohol at 80°C (forced-draft oven) and store over concentrated H,SO, in vacua. Determine residual moisture on an aliquot by drying at 110°C. 2. Loss of tryptophan in the samples by oxidation or photolysis on prolonged storage, especially in the presence of moisture, may be minimized by refrigeration in air-tight brown bottles. Peptide-bound tryptophan, being more reactive (see Results and Discussion) may be more susceptible to destruction. Loss of tryptophan presumably is minimized during grinding by water-cooling. 3. Proper choice of reaction conditions and butanol extraction of sample minimize, if not eliminate the interference of grain pigments that posed serious difficulties with the Opienska-Blauth method (17). 4. To prevent scattering and contamination of samples, cover them with several layers of tissue paper tied securely to the tube before drying the sample with a vacuum pump. 5. The torsion balance is ideal for rapid weighing of several samples for extraction. Time is saved if one sample is extracted while the next one is being weighed. Accurate weighing is not necessary since the nitrogen of the extract is determined. Formation of clumps which are difficult to disperse is prevented by adding the sample to the NaOH solution instead of the reverse. The recommended weights of sample correspond to between 20 and 40 ,ug of tryptophan. The maximum tryptophan value which still conforms to Beer and Lambert’s law does not exceed 60 pg (Results and Discussion). Between 250 and 300 mg may be required for the relaZThe follows:
Jones (20) nitrogen-protein conversion factors (g of N/l00 g of protein) Barley, 17.2; corn, 16.0: oats, 17.2: rice, 16.8: rye, 17.1; wheat 17.1.
are as
RAPID
TRYPTOPHAN
METHOD
FOR
CEREALS
211
tively low-tryptophan grains, such as corn and barley. Interferences are minimized with smaller sample weights. 6. With 0.075 N NaOH, the protein extraction efficiency, of samples stored in the refrigerator for several months is 90% or greater. The value for freshly ground samples generally is 98% or greater. 7. Poor reproducibility of results may be obtained if the extract is allowed to flow down the side of the test tube during pipetting. The reason is that an indefinite but significant amount of protein may remain on the side of the tube which, obviously, may not be reacted equally with the rest of the sample, if at all. 8. Serious browning or even charring may result if H,SO, is added first to the sample, instead of acetic acid. The large amount of heat produced when H&SO, is added to the alkaline extract may destroy most of the tryptophan. 9. Proceed to determine the absorbance of the samples while the standard solution is still developing in the water bath. For each batch of reagents, include a tryptophan standard for each daily run (see Note 11). For precision, immerse at least 3/4 of the tube during heating to insure equal heating conditions for any liquid on the side of the tube. 10. Factors such as inherent sample turbidity, which is not removed by filtration or centrifugation, may result in still significant blank values in spite of butanol extraction. When extracts are clear and almost colorless, blank corrections may be omitted. Even though some samples may develop color at 25°C faster (e.g., wheat) than others, the time lag of 5-6 min, before a significant absorbance due to the chromophore can be detected, permits the determination of the sample blank. Samples are cooled in ice water to delay color development during removal of bubbles. However, readings must be done with samples at almost room temperature. Only one sample blank at a time may be processed for absorbance reading within 5 min without use of a flow cell. Otherwise, 2-3 samples are possible. Since the test solutions are more viscous than water, bubbles can only be effectively removed by suction or by heating. Very small bubbles which are invisible may coalesce during absorbance reading. No bubbles are present in the test solutions heated at 60°C. Thus, the unheated sample blanks must be suctioned thoroughly to insure complete removal of bubbles. 11. A standard curve may be plotted with each fresh batch of reagents. However, it is preferable to include a standard tryptophan solution with each run as an internal check for possible variation in concentrations of reagents or reaction conditions (see Results and Discussion).
212
JOSE
MADRID
CONCON
TABLE TRYPTOPHAN
CONTENT
SPECIES
OF CEREAL AND
Cereal grams Barley Missouri B 415 Knob CI 11910 McNair 601 Corn Tolima 377 Brazil 70 Opaque-2 Oats Commercial Mix Wyndmere Rice BPI-76- 1d IR-8 LPd IR-8 HP@ Wheat Hard red Winter wheat Azteca 67 Unbleached Floure Rye Tetra Petcus Michigan rye
THE
OF PROTEINS GRAINS PROCEDURE
1
FROM
DIFFERENT
AS OBTAINED OF SPIES
WITH AND
VARIETIES THE
PRESENT
OF VARIOUS METHOD
(6.9)
CHAMBERS
Tryptophan, g/l00 g of proteins
Total nitrogen (%I”
Total nitrogen extracted m
Present methodD,C
Spies and ChambersbSc
1.63 2.40 2.47
98.3 100.7 97.8
1.71 1.39 1.49
1.45 1.47 1.29
1.79 1.12 1.11
92.22 100.7 105.6
0.41 0.52 0.88
0.32 0.47 0.75
2.49 2.29
100.0 99.6
1.02 1.20
1.00 1.22
2.52 1.19 1.62
97.5 90.0 88.3
1.42 1.44 I .59
1.42 1.36 1.43
2.89 2.88
97.7 99.6
1.10 1.18
1.04 1.36
2.27
103.6
0.86
0.81
1.79 2.17
95.8 92.9
0.99 I .03
0.83 0.85
u “As-is” basis. b Average of duplicate determinations. Average difference of individual values from the corresponding mean is 0.01. c Nitrogen-protein conversion factor: 16 g of N/100 g of protein, except rice for which 16.8 g was used. d Samples supplied by Dr. B. Juliano, International Rice Research Institute, Los Baiios, Laguna, Philippines. p Unknown variety.
RESULTS AND
DISCUSSION
The tryptophan content of the proteins of several varieties of different species of cereal grains are shown in Table 1. The values obtained with this method are compared with those of the method of Spies and Chambers (5,6,9). Agreement between the two methods is highly satis-
RAPID
TRYPTOPHAN
METHOD
FOR
CEREALS
213
factory. Values recorded in Table 1 are very precise. The average difference between the mean and the corresponding individual values of each duplicate is about 0.03 units. Except for corn, samples analyzed with the Spies and Chambers method were first extracted with 0.075 N NaOH. The extract (0.50 ml) was treated with 0.5 ml of Pronase solution [4 mg of PronaselO.5 ml of phosphate buffer, 0.1 M, pH 4.4 (Pronase activity, 45,000 PUK units/g, Calbiochem, Los Angeles, CA 90054)], and digested for 48 hr at 40°C with a few drops of toluene as preservative. The final mixture had a pH between 7.5 and 8.5, the optimum range for Pronase activity (9,21). Ethanol interference (13) precluded the use of the extraction technique of Concon (14) for corn proteins in conjunction with the Spies and Chambers (5,6,9) method. Instead, corn powder was digested directly with Pronase for 48 hr at 4O”C, based on the recommendation of Spies (9). [About 100 mg of corn/ml of Pronase solution (2 mg of crude enzyme/ml of 0.1 M phosphate buffer, pH 7.5).] Between 93 and 103% of corn nitrogen was solubilized; the digest did not give any abnormal colors with the reagents. For the sample blank, NaNO, was added first to the sample before the p-dimethylaminobenzaldehyde (DAB)-H,SO, solution. The absorbance was read immediately. Many samples developed between 20 and 40% of the maximum color intensity obtainable in the oxidation with NaNO, during the 18-hr incubation with DAB and H&SO,. This suggests the presence of unspecified oxidants in cereal grains. Conformance
to Beer and Lambert’s
Law
As shown in Fig. 1, the glyoxylate reaction under the conditions in this method conforms very well to the Beer and Lambert’s law only up to about 60 pg of free or cereal protein-bound tryptophan. There is a gradual deviation above this value so that Eq. [l] is no longer valid. The deviation appears to be a property of the glyoxylate reaction and not an indication of the exhaustion of the available glyoxylate since, from 20- 100 pg of tryptophan, the glyoxylate reaction under the conditions in this method can be more precisely represented by Eq. [ 31 instead of the Beer and Lambert’s law. Log T = k/l + log b,
[31
where T = tryptophan concentration; A = absorbance; k = a proportionality constant, and b = the tryptophan value at zero absorbance, in this case between 11 and 12 pg. The lack of correlation of the experimental results below 20 pg in this case may be ascribed to experimental error which is greatly exaggerated by a logarithmic plot. Thus, Eq. 3 may be used for calculating tryptophan values between 20 and 100 pg.
214
JOSE MADRID
CONCON
.240 s 2 2 8 z
.200
0
CORN
-O-
OATS
0
W"EA1P
-160
TRYPTOPHAN
,120 ,120
.OPO
.040 .020 10
20
40
60 ),g
80
100
120
TRYPTOPHAN
FIG. 1. A Beer and Lambert’s plot of the glyoxylate reaction with tryptophan and various unhydrolyzed cereal proteins.
But because of its simplicity, calculations based on the Beer and Lambert’s law (Eq. [l] is preferable for tryptophan values below 60 pg. Sensitivity, Accuracy and Precision, and Specificity of the Method The minimum amount of tryptophan that may be determined reliably in this method is about 6-7 pg (Fig. l), provided the sample blank is negligible. An ultramicroadaptation would lower this value to at least 1 t-a* The accuracy of this method may be compared only with the Spies and Chambers method (6,9). The highly satisfactory agreement of the results obtained with these methods has already been mentioned. A possible source of error in the Spies and Chambers method (6,9) is the destruction of tryptophan during the prolonged incubation in 19 N
RAPID
TRYPTOPHAN
METHOD
FOR
CEREALS
215
H,SO, (22). However, free tryptophan was shown to be remarkably stable in 19 N H,SO, (5), and Pronase digestion, very likely, liberates all the tryptophan residues in cereal proteins (20). The Spies and Chambers method has been used satisfactorily with various food products (12,23), with tryptophan values in satisfactory agreement with the results of microbiological assays (23). The slight browning of solutions observed during incubation with H,SO, is minimized if not eliminated by NaNO,. This browning may be a source of positive error in the Spies and Chambers method. The high precision of the present method has already been mentioned. Individual results for the same sample obtained on different days differed by no more than 0.03 units. Other indoles which also react with glyoxylate in the presence of H,SO, (5,24) are not present in cereal grain extracts in significant amounts, if at all. No other compounds in cereal grains are known to react significantly with glyoxylate. And unlike in the Spies and Chambers method (5,6,9), ethanol does not interfere. With the sample blank correction, the present method is highly specific for tryptophan in cereal grains. The Tryptophan Chromophore and Its Stability Normally, the tryptophan chromophore gives a violet solution in acid medium. But occasionally, as in rye, barley, and, sometimes, wheat, the solution is red-violet or magenta, without affecting the A,,,. The reddish hue of the solution, in this case, may be due to certain grain pigments with negligible absorption at 545 nm. The color of the solution is stable even after several hours in bright laboratory light. However, light may have a slight (about 4%) colordeepening effect during development. A similar effect of light was also observed by Spies and Chambers (7) with DAB. Thus, because of possible unequal illumination, the samples were covered during color development for maximum precision. The tryptophan chromophore is stable even after 1 hr at 60°C. Only one barley sample showed a slight decrease within this time. However, above 70°C the chromophore is gradually destroyed at different rates in different samples. Barley and corn showed faster rates of destruction than the other grains. And at 100°C 80% or more of the chromophore in most samples may be destroyed in 10 min. Effects of Sulfuric Acid and Water Concentrations As seen in the following schemes, H,SO, functions both as a proton donor and dehydrating agent in the formation of the tryptophan chromophore (Scheme I) and of glyoxylate from acetic acid (Scheme II).
216
JOSE MADRID Scheme
I
[Formation
mtyg Scheme
II
of
the
Tryptophan
(Formation
(4,25)1
of
2
OOH
Glyoxylate)
H2S04
*
) (01,
Chromophore
*cg-;ooH ;;-TTH < ,H--
Fe +++ CH3COOH
CONCON
H+
<-
(OH)2-CH-COOH glyoxylic hemihydrate
O=CH-COOH S20
acid
glyoxylic
acid
In cereal proteins (represented by rice and wheat), color development, expressed as percent of the maximum color obtained under the conditions chosen for this method, is a direct and inverse function of the H,SO, and H,O concentrations, respectively (Fig. 2A and B). For the data in Fig. 2, a IO-min reaction time was chosen since differences in reaction rates are more pronounced in the initial stages of the reaction. 160
160
4
6
E
10
12
NORMALITY OF H2S04 IN THE FINAL MIXTURE
MOLARITY OF II20 IN THE FINAL MIXT”,?Z?
FIG. 2. Effect of (A) H,SO, and (B) H,O concentrations on the color development of the glyoxylate reaction with free and cereal protein-bound tryptophan.
RAPID
TRYPTOPHAN
METHOD
FOR
CEREALS
217
The steep slope of these curves (3.5) represents a 17% increase in color intensity for each 1 N increment in acid concentration. Thus, color development is highly sensitive to acid concentration. The color development of free tryptophan under the same conditions as with protein samples gives a sigmoid-shaped curve (Fig. 2) which can be expressed semilogarithmically as in Eq. [4]. LogA=bS-CC,
[41
where A = absorbance, S = acid or water concentration, C = the concentration at zero absorbance; and b = slope of the curve which is negative when S is the water concentration. The sigmoid curve, in contrast to the linear cereal-protein curves, suggests the possible influence of the free amino or carboxylic groups in the color development. Experimentally, Eq. [4] is valid only up to 10 N H&SO,, since, above this value, significant destruction of tryptophan occurs which is accompanied by increased browning, in the case of the cereal extracts, or increased cloudiness, in the case of the standard. Thus, 10 N H,SO, is the practical limit to which acid concentration can be increased for the purpose of increasing the sensitivity of the method. In rye and barley, browning and other-color formations occur even at a much lower acid concentration. [These grains produced red solutions in the OpienskaBlauth method ( 17)]. The primary function of H,SO, in the reaction is probably as a dehydrating agent rather than as a proton donor. This function is probably aptly illustrated in Fig. 2B which shows a rapid decrease in color with increasing water concentration, and a zero absorbance at 20 M H,O. The sensitivity of the glyoxylate reaction to water concentration was also observed previously by others (15, 17, 22). The dehydration of the intermediate carbinol in Scheme I may be influenced by the water concentration, not only by mass action but also by its effect on the dehydrating capacity of H,SO,. In addition, increased water concentration may also favor the formation of a very stable glyoxylate hemihydrate (26) which is expected to be less reactive to tryptophan. The Rate of Color Formation
In Fig. 3, one notes that, between 60 and 70°C only acid concentration among the factors studied can affect the time maximum color is attained with free tryptophan. Higher temperatures cannot be tested because of the destruction of tryptophan or its chromophore. Copper, whose color-enhancing effect was noted by Winkler (27), has no effect on the time of maximum color development. On the contrary, its use in
218
JOSE
MADRID
CONCON
.22,
.20.
&iYTrzlo4, 7o”c
.18
Pd ,
.I6
.
8.6
N i12S04,
GOOC +
COPPER
-
d II
0
f3.G N H2S”4,
60°C
0
7.9
GODC
/cfi
.14 _
El H7S04,
e 2Fj
. 12.
5 .10.-
.08 -
.OG _
.04
-
MINUTES
FIG. 3. The rate of color development of the glyoxylate reaction with free tryptophan under different conditions. Inset: The rate of color development of the glyoxylate reaction with different cereal proteins at 60°C.
the present method is precluded since it promotes intense browning of solutions in proportion to the amount of metal present. Among the cereal grains (Fig. 3, inset), only oats show a slight delay in the attainment of maximum color, which, for most grains, occurs after 30 min at 60°C. ACKNOWLEDGMENTS I thank Dr. R. J. Dimler, Northern Regional Research Laboratory, USDA, Peoria, IL; Dr. E. L. Kendrick, Cereal Crops Research Branch, Plant Industry Station, USDA, Beltsville, MD; and Dr. L. V. Packett, Department of Nutrition and Food Science, University of Kentucky, Lexington, KY 40506, for making this work possible; and especially Mr. C. H. Van Etten, Northern Regional Research Laboratory, Peoria, IL, for his valuable assistance and for providing the samples: I also thank many generous individuals for their efforts in providing most of the samples and others, for their technical and secretarial assis-
RAPID
TRYPTOPHAN
METHOD
FOR
CEREALS
219
tance. My thanks also to Mr. Jerry Reisig for his inadvertent assistance which helped in speeding up the work.
REFERENCES 1. 2. 3. 4. 5. 6.
7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
Matsubara, H., and Sasaki, R. M. (1969) Biochem. Biophys. Res. Commun. 35, 175. Penke, B.. Ferenczi, R.. and Kovacs, K. (1974) Anal. Biochem. 60,45. Liu, T. Y., and Chang, Y. H. (1971) J. Biol. Chem. 246, 2842. Friedman, M., and Finley, J. W. ( 197 1) .I. Agr. Food Chem. 19, 626. Spies, J. R., and Chambers, D. C. (1948) Anal. Chem. 20, 30. Spies, J. R., and Chambers, D. C. ( 1949) Anal. Chem. 21, 1249. Spies, J. R., and Chambers, D. C. (1950) Anal. Chem. 22, 1209. Spies, J. R. (1950) Anal. Chem. 22, 1447. Spies, J. R. (1967) Anal. Chem. 39, 1412. Spies, J. R. (1968)5. Agr. Food Chem. 16, 514. Vangala, R. R., and Menden, E. (1970) Z. Lebensm. Untersuch. Forsch. 142, 195. Lombard, J. H., and Delange, D. J. (1965) Anal. Biochem. 10, 260. Concon, J. M. (1975) Anal. Biochem, in press. Concon, J. M. (1973) Anal. Biochem. 55, 563. Fischl, J. (1960) J. Biol. Chem. 235, 999. Adamkiewicz, A. (1875) Berichte 8, 1 11. Opienska-Blauth, J., Charenzinski. M., Berbec, H. (1963) Anal. Biochem. 6, 69. Concon, J. M. (1975) Anal. Biochem., in press. Concon, J. M., and Soltess. D. (1973) Anal. Biochem. 53, 35. Jones, D. B. (1931) U.S.D.A. Circular No. 183, Washington, DC. Nomoto, M.. Narahashi, Y., and Murakami, M. (1960) J. B&hem. (Tokyo) 48, 593. Inglis, A. S., and Leaver, I. H. (1964) Anal. Biochem. 7, 10. Food and Agricultural Organization of the United Nations (1970) Ammo Acid Contents of Foods and Biological Data on Proteins, Rome. Remers, W. A. (1972) in Indoles Part I (Houlihan, W. J., ed.), p. 1, Wiley-Interscience, New York. Lugg, J. W. H. (1938) Biochem. J. 32, 775. Fieser, L. T., and Fieser, M. (1956) Organic Chemistry, D. C. Heath, Boston. Winkler, S. (1934) Z. Physiol. Chem. 228, 50.